Overview of the work:
The initial phases of the project focused on two points 1) spatial/temporal profiling of Nalcn expression body-wide and 2) characterization of the conditional Nalcn allele mouse model. In order to determine the spatial/temporal profiling of Nalcn expression, I evaluated a variety of molecular (qRT-PCR) and histological approaches (in situ hybridization and immunohistochemistry) to accurately identify Nalcn expressing cells in E9.5-E18 and postnatal D1 and D60 tissue samples. These experiments lead to the development of new in situ hybridization probes, identification of numerous inaccurate antibodies and one reliably validated antibody. Using these tools, we determined spatial/temporal profiling of Nalcn. We observed similar results to what was described in the literature, but we were able to further characterize the localization of Nalcn to a variety of stem cells in multiple tissues throughout body including stomach, small intestine, colon, brain and skin.
The next steps of our experiments involved devising a series of strategic genetic mouse model crosses to determine the role of Nalcn in normal development and tumourigenesis. We used the Prominin-1 driven Cre recombinase mouse model to delete Nalcn in stem cells body-wide. We combined this with a conditional reporter allele, ZSGreen, to identify targeted cells histologically. For understanding the role of Nalcn in normal development, we generated experimental (Prom1C-L/+;R26ZSGreen;Nalcnf/+ , Prom1C-L/+;R26ZSGreen;Nalcnf/f) and control (Prom1C-L/+;R26ZSGreen) mouse cohorts. To induce Nalcn loss, animals received two intraperitoneal injections of tamoxifen at postnatal time points P1/2 (development) or P60/61 (homeostasis) then aged and monitored for phenotypic alterations. Additionally, we crossed known oncogenes (KRASG12D) and tumour suppressor genes (Trp53f/f) to the conditional allele to asses the impact on tumour formation. Our preliminary data indicate Prom1C-L;KRASG12D;Trp53f/f mice develop gastric cancers from Prom1+ malignant stem cells. Tamoxifen injected animals (neonatal or adult) are being aged and sacrificed, tissues harvested at designated time points or at first signs of illness for histological analysis and transcriptomic profiling.
Our preliminary histological analysis of tissues from Nalcn modified animals at several time points post tamoxifen induction (30d, 90d, 180d, 270d, 365d) in our developmental models demonstrated that Nalcn loss alone has an important role in normal cellular development and a tumour suppressive role for many tissue types. We observed maldifferentiation in the GI tract, fibrosis in several tissues body-wide and tumour formation. Moreover, reduction in Nalcn expression cell autonomously significantly increased non-cell autonomous immune infiltration and inflammation in several tissues including the GI tract. In our tumour background models, Nalcn loss has enhanced tumour penetrance, metastasis and increases inflammation. We are currently carrying out additional in depth histological profiling of these tissues to better resolve the phenotypes associated with Nalcn loss.
In order to better understand the mechanism of Nalcn in the GI tract, we carried out transcriptomic profiling of GI tract tissues from 9-month old animals with no overt histological phenotype. Analysis of the stomach data identified a pro-inflammatory signature and a panel of genes significantly differentially expressed in human gastric cancer. Further in depth analysis is required and we are expanding the transcriptomic profiling to early stages and additional tissues (e.g. kidney, epididymis).
Exploitation and dissemination:
This work has presented and discussed at variety of venues including the CRUK Cambridge Institute seminar series, the Cambridge Cancer Centre Symposium, the International Society for Stem Cell Research, Cell Symposia Cancer, Inflammation and Immunity and the Cell Symposia.
